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Ivyspring International Publisher Journal of Cancer 2020; 11(1): 190-198. doi: 10.7150/jca.36954 Research Paper C-MYC, BCL2 and BCL6 Translocation in B-cell Non-Hodgkin Lymphoma Cases Dayang Sharyati Datu Abdul Salam1, Ei Ei Thit2, Siew Hoon Teoh1, Soo Yong Tan3, Suat Cheng Peh4, Shiau-Chuen Cheah1

1. Faculty of Medicine and Health Sciences, UCSI University, Kuala Lumpur, Malaysia. 2. Advanced Molecular Pathology Laboratory, SingHealth Tissue Repository, Singapore. 3. National University Hospital, Singapore. 4. Sunway University Hospital, Selangor, Malaysia.

 Corresponding author: [email protected]

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2019.05.23; Accepted: 2019.09.23; Published: 2020.01.01

Abstract C-MYC, BCL2 and BCL6 are the most commonly involved in B-Cell lymphomas. Translocations of these oncogenes are associated with an aggressive clinical course. This study aims to elucidate the patterns of BCL6, BCL2 and C-MYC aberrations among Malaysian B-cell Non-Hodgkin Lymphoma (NHL) using fluorescence in situ hybridization (FISH). Eighty-one B-cell NHL tissue blocks were retrieved between the year 2011 to 2015 and investigated using immunohistochemistry and interphase FISH dual colour break-apart probes of BCL2, BCL6, C-MYC and IgH. A significant difference was detected between the nodal and extranodal sites in all the BCL2 (p=0.01), C-MYC (p=0.03) and IgH (p=0.006) cases except for BCL6 (p=0.2). Our study showed that BCL6 had the highest gene translocation while BCL2/BCL6 had the most mixed aberrations of gain copies and translocation, however no mixed aberrations of gain copies and translocation was found in C-MYC. None of the mixed gain copies and translocation was found in any of the germinal centre B-cell (GCB) subtype of Diffuse Large B-cell Lymphoma, however, five were found in BCL6 and IgH gene in the non-GCB subtype; while mixed gain copies and translocation cases of BCL2 gene was found in the Follicular Lymphoma cases only. The study found interesting findings of BCL2, C-MYC and IgH gene aberrations between nodal and extranodal sites. This information might benefit future study in predicting prognosis and determine effective therapeutic strategies in the multi-ethnic populations of Malaysia as well as the Asian population.

Key words: B-cell Non-Hodgkin Lymphoma, C-MYC, BCL2, BCL6, gene abberation, FISH

Introduction There has been an intense upsurge worldwide diagnosing cancer cell type, thus leads to lesser for Non-Hodgkin lymphoma (NHL) incidence misdiagnosis cases of NHL as well as better approximately 2-4% annually since the last seventy classification of gray-zone lymphomas [4]. In years [1]. Based on the statistics presented, it was in comparison to western countries, Asian countries accordance to the study done by Devesa and Fears [2] have lower median age of NHL. The representative whereby western countries have the highest increased median age of patient in western countries was 68 observed. Both males and females have seen an years old [5]. However, the median age of NHL increased in all age groups however racial differences patient in Asian countries such as Iran, Korea and was observed in age-specific incidence curves up until Taiwan are between 50-55 years old [6-8]. In the the age of 45 and 35 for male and female respectively Global Cancer Facts 3rd Edition [9], the global trend [3]. The reasons for the increase include wider use of suggested that the increase of NHL cases in most immunohistochemical method for screening and developed countries reflects a true increase in disease

http://www.jcancer.org Journal of Cancer 2020, Vol. 11 191 occurrence. However, improvements in diagnostic in cell cycle control, proliferation and differentiation, methods and modification of classification may also apoptosis, and DNA damage response. Loss of play a role in the increase of NHL cases [10]. normal control mechanisms regulating BCL6 Double-hit translocation refer to translocation of expression causes lymphoproliferative disease, C-MYC with another concurrent breakpoints such as resembling DLBCL. BCL6 translocation, t(3q27), is BCL2, BCL6, BCL1 or BCL3. Triple-hit translocation responsible for up to 35% of DLBCL cases, the largest consists of C-MYC, BCL2 and BCL6. Currently, these compared to other DLBCL gene translocations [17]. gene translocations do not have any baseline Deregulation of BCL6 located on 3 is characteristics aside from the genetic profile that can believed to contribute to malignant transformation in be confirmed through various genetic tests such as germinal center–derived B cells (GCB). Detection of fluorescence in situ hybridisation (FISH). Also, point mutations of the regulatory region of the BCL6 morphologically they are difficult to distinguish and gene have been frequently found in GCB and post– are usually unclassifiable B-cell lymphoma of GCB lymphomas, including FL, DLBCL, and BL [18]. intermediate characteristic between aggressive The genetic aberrations that are representative of lymphoma, Diffuse Large B-cell Lymphoma (DLBCL), specific subtypes of NHL mentioned previously can and very aggressive lymphoma, Burkitt Lymphoma be identified by genetic diagnosis. Particularly, the (BL). use of FISH studies to diagnose NHL is becoming C-MYC translocation of t(8;14)(q24;q32) usually increasingly important. Interphase FISH is the most deregulate C-MYC expression. Rearrangement of the commonly applied technique for the demonstration of C-MYC gene situated on chromosome 8 next to the gene translocation, as it can be applied on paraffin IgH gene, or lambda (λ) and kappa (κ) light chain embedded tissue, enabling studies on archival genes subsequently caused upregulation of gene material. In comparison to immunohistochemical expression commonly observed in BL. In normal cells, staining, interphase FISH are able to visualize gene C-MYC acts as a transcriptional regulator involved expression pattern and can provide spatial and largely with cell cycle progression (from G1 to S temporal information on understanding gene function phase) and the inhibition of terminal differentiation. which immunohistochemistry are not able to provide. Over-expression in normal cells sensitizes the cell to a Hence, the study aims to characterize the patterns of variety of apoptotic triggers which leads the cell to BCL6, BCL2 and C-MYC gene aberrations in resist cell death ultimately result in cancer. C-MYC Malaysian B-cell NHL using interphase FISH. gene rearrangement is reported to be rare occurrence in Follicular Lymphoma (FL). However, it was Patients and Methods observed in 7-15% of de novo DLBCL [11] and 8% of Cases selection post-transformation DLBCL [12]. Several studies reported [13,14] the presence of C-MYC gene A total of 81 B-cell Non-Hodgkin Lymphoma rearrangement upon transformation. Hence, it is (NHL) were retrieved from a private hospital in highly probable that C-MYC plays a vital role in Penisular Malaysia laboratory, the Pantai Premier transformation of high grade B-cell malignancies. Pathology Malaysia archives between the years 2011 BCL2 translocation, t(14;18)(q32;q21), is observed to 2015. The demographic data of these patients, in about 15-20% of DLBCL cases and approximately in immunohistochemistry slides and tissue blocks were 80-90% of FL cases [15]. BCL2 came from the BCL obtained from the Pantai Premier Pathology database family which has a crucial role in cell by the referring clinician. All the B-cell NHL were apoptosis. BCL2 function as a pro-survival , sectioned at 3 µm for immunohistochemical staining represses apoptotic cell death, and protects cell from a using a panel of monoclonal and polyclonal wide range of cytotoxic insults including cytokine according to Pantai Premier Pathology’s deprivation, ultraviolet irradiation. Deregulation of standard operating procedure. For the DLBCL BCL2 leads to over-expression of BCL2, making the classification of GCB and non-GCB subtype, a panel of cell to resist cell death. In contrast, BCL2 inhibition three antigens namely CD10, BCL6 and MUM1 was eliminate cell survival advantage and allow apoptosis used according to Hans Criteria [19]. to occur. Similar to C-MYC translocation characteristic Morphology in DLBCL, chromosomal rearrangement that mix the The materials were cut into 4 µm thick sections BCL2 gene situated on chromosome 18 involving the and stained with haematoxylin-eosin for histologic IgH gene deregulates the BCL2 expression [16]. evaluation. All specimens were reviewed by a Coming from the same BCL family proteins, pathologist (PSC) for confirmation and classification BCL6 is essential for the development of germinal according to the 2008 WHO Classification of tumours centre in B-cells. It acts as a transcriptional repressor of haematopoietic and lymphoid tissues [4] using

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Leica DM500 light microscope (Leica Microsystems, Lymphoid Tissue (MALT) cases (1%), 1 Mediastinal Switzerland) graded reticule at x10 and x40 B-cell Lymphoma case (1%) and 1 magnification. DLBCL-Plasmablastic Lymphoma case (1%). In terms of lymphoma sites, B-cell lymphoma originating from Interphase FISH and scoring nodal sites was 42 (52%) and extranodal sites was 39 A total of 81 cases were examined using four (48%). The most common extranodal sites are interphase FISH dual colour break-apart probes gastrointestinal (12 cases, 31%) and Waldeyer’s ring (8 namely BCL2/18q21, BCL6/3q27, C-MYC/8q24 and cases, 21%) and 19 others for example in the brain or IgH/14q32 (Dako, Denmark). Haematoxylin-eosin spleen. stained tissue sections from representative formalin-fixed, paraffin-embedded tissue blocks were Table 1. Demographic and clinical characteristic of 81 patients used to define the tumour areas. FISH analysis was with B-Cell NHL performed on 2 µm tissue sections using FISH DNA break-apart probes and FISH Ancillary Kit (Vysis N (%) Gender probe, Abbott, United States). The method was Male 42 (52) carried out at Advanced Molecular Pathology Female 39 (48) Laboratory (AMPL), SingHealth, Singapore according Age to AMPL’s standard operating procedure. Briefly, the <60 years 48 (59) ≥60 years 33 (41) tissue slide was de-paraffinised and rehydrated prior Ethnic to the procedure. After digestion with pepsin Malay 23 (28) solution, FISH DNA Probe was added to the tissue Chinese 42 (52) Indian 4 (5) sections and hybridized for three nights followed by Others 12 (15) stringent wash. Assessment of signals was performed Sites using Zeiss Axioimager Fluorescent microscope Nodal 42 (52) (Germany) with the Metasystem’s ISIS Metafer Extranodal 39 (48) software (Germany) at x63 magnification. The cases performed depend on the availability of tissue block Prevalence of BCL2 / BCL6 / C-MYC / IgH gene hence the total cases for each gene were different. The aberrations FISH analyses were divided into normal, translocated A summary of all the FISH prevalence are given (>20% of the whole tumour population), gain copies in Table 2. Out of the 81 cases, only 73 cases had (>50% of the whole tumour population) and both successful BCL2 FISH assay, 67 patients had translocated and gain copies category. successful BCL6 FISH assay, 72 successful FISH assay Statistics were collected for C-MYC and 64 cases had successful IgH FISH assay. In DLBCL cases, BCL6 and IgH gene Statistical analysis was completed using aberrations recorded the highest with 19 and 20 cases GraphPad Prism software (version 5.0; GraphPad respectively. In terms of DLBCL cell-of-origin (COO) Software Inc., San Diego, CA). χ2 or Fisher’s exact tests classification, there was no significant difference were used for categorical parameters to identify between the gene aberrations to GCB and non-GCB significant relationships between variables. P value < type except for BCL6 (p=0.03*). BCL2 and IgH were the 0.05 was considered statistically significant. highest gene aberrations in FL cases with 6 and 7 cases Results respectively. One BCL2 gene aberrations was found in MZL and one C-MYC and IgH gene aberrations were Patients’ Characteristics found in a BL case. Although female had slightly The patients’ demographic and clinical higher C-MYC and IgH gene aberrations compared to characteristics are summarized in Table 1. Among the male, there was no significant difference found 81 patients, there were 42 males (51.9%) and 39 between them. While, Chinese had the highest gene females (48.1%) in this study. The male:female ratio is aberrations in all the FISH BCL2, BCL6, C-MYC and 1.08:1 and age ranged from 10-92 (median age of 55.8). IgH assays although there was no significant The ethnic distribution were as follow: 23 Malays difference found among the Malaysian ethnics. (28.4%), 42 Chinese (51.9%), 4 Indians (4.9%) and 12 Significant difference was detected between the nodal Others consisted of Bumiputera and foreigners with and extranodal sites in all the BCL2 (p=0.01**), C-MYC undetermined country of origin (14.8%). There were (p=0.03*) and IgH (p=0.006**) except for BCL6 (p=0.2), 57 DLBCL cases (70%), 15 FL cases (19%), 1 BL case although higher BCL6 gene aberrations were found in (1%), 1 DLBCL-BL like case (1%), 4 Marginal Zone the nodal compared to extranodal sites. Lymphoma (MZL) case (5%), 1 Mucosa-Associated

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Table 2. Prevalence of BCL2 / BCL6 / C-MYC / IgH gene aberrations in B-Cell NHL patients

BCL2 (n= 73) BCL6 (n= 67) C-MYC (n= 72) IgH (n= 64) Normal Aberrations Normal Aberrations Normal Aberrations Normal Aberrations N (%) N (%) N (%) N (%) N (%) N (%) N (%) N (%) B-cell NHL p = 0.22 p = 0.06 p = 0.87 p = 0.06 DLBCL 35 (48) 16 (22) 28 (42) 19 (28) 41 (57) 9 (13) 24 (38) 20 (31) FL 7 (10) 6 (8) 7 (10) 4 (6) 11 (15) 2 (3) 4 (6) 7 (11) Others 8 (11) 1 (1) 9 (13) 0 (0) 8 (11) 1 (1) 8 (13) 1 (2) Gender p = 1.0 p = 0.8 p = 0.54 p = 0.46 Male 25 (34) 12 (16) 21 (31) 12 (18) 32 (44) 5 (7) 19 (30) 12 (19) Female 25 (34) 11 (15) 23 (34) 11 (16) 28 (39) 7 (10) 17 (27) 16 (25) DLBCL COO p = 0.47 (n=51) p = 0.03* (n=47) p = 0.43 (n=50) p = 0.73 (n=44) GCB 9 (18) 2 (4) 10 (21) 1 (2) 11 (22) 1 (2) 6 (14) 4 (9) Non-GCB 26 (51) 14 (28) 18 (38) 18 (38) 30 (60) 8 (16) 18 (41) 16 (36) Site p = 0.01* p = 0.2 p = 0.03* p = 0.006* Nodal 21 (29) 17 (23) 21 (31) 15 (22) 29 (40) 10 (14) 14 (22) 21 (33) Extranodal 29 (40) 6 (8) 23 (34) 8 (12) 31 (43) 2 (3) 22 (34) 7 (11) Ethnic p = 0.54 p = 0.78 p = 0.49 p = 0.17 Malay 12 (16) 7 (10) 13 (19) 6 (9) 15 (21) 4 (6) 13 (20) 5 (8) Chinese 26 (36) 13 (18) 21 (31) 13 (19) 31 (43) 7 (10) 18 (28) 15 (23) Others 12 (16) 3 (4) 10 (15) 4 (6) 14 (19) 1 (1) 5 (8) 8 (13) COO, cell-of-origin

Table 3. FISH analysis of BCL2, BCL6, C-MYC and IgH gene only 2 (13.3%) C-MYC gain cases. In addition, 1 aberrations C-MYC translocated case was found in BL. While for DLBCL, n=57 FL, n=15 Others, n=9 the IgH gene abnormalities, among the 57 DLBCL N (%) N (%) N (%) cases, there were 1 (1.8%) IgH gain, 17 (29.8%) IgH BCL-2 Gain 13 (22.8%) 0 1 (11.1%) *MZL translocated and 2 (3.5%) IgH translocated and gain Translocated 3 (5.3%) 4 (26.7%) 0 cases. Apart from that, FL had only 7 (26.7%) IgH Translocated & Gain 0 2 (13.3%) 0 translocated cases. BCL-6 Gain 6 (10.5%) 1 (6.7%) 0 Double and triple aberrations among BCL2, Translocated 10 (17.5%) 3 (20.0%) 0 BCL6 and C-MYC Translocated & Gain 3 (5.3%) 0 0 C-MYC There were 16 cases found to have concurrent Gain 5 (8.8%) 2 (13.3%) 0 genetic aberrations through FISH analyses on BCL2, Translocated 4 (7.0%) 0 1 (11.1%) *BL BCL6 C-MYC Translocated & Gain 0 0 0 and . These cases were investigated IgH further by doing comparison of their immune- Gain 1 (1.8%) 0 (0.0%) 0 phenotype and genetic features as shown in Table 4 Translocated 17 (29.8%) 7 (46.7%) 1 (11.1%) *BL while summary of the gene aberrations are shown in Translocated & Gain 2 (3.5%) 0 0 Table 5. Of the 16 cases, 13 were DLBCL and 3 were MZL, Marginal Zone Lymphoma; BL, Burkitt Lymphoma FL cases. There was equal proportion of male and

female however higher aberrations were found in the Analyses of BCL2 / BCL6 / C-MYC and IgH gene nodal sites (12 cases) than extranodal sites (4 cases). aberrations One double-hit lymphoma of BCL6/C-MYC was Table 3 summarised the FISH analysis of BCL2, established in DLBCL Case 6 (Fig 1), however both of BCL6, C-MYC and IgH gene aberrations according to the BCL6/C-MYC protein expression showed less NHL subtypes. Among the 57 DLBCL cases, there than 50% and 40% respectively with Ki67 of 40%. were 13 BCL2 gain (22.8%) and 3 (5.3%) BCL2 Apart from that, two translocation of BCL2/BCL6 translocated cases. While, FL had 4 (26.7%) BCL2 (Case 3 and Case 14) cases were found with the Case translocated cases and 2 (13.3%) BCL2 translocated 14 having additional 3-4 gain copies of C-MYC. The and gain cases. In addition, 1 BCL2 gain case was corresponding BCL2/BCL6 co-expression show posi- found in MZL. On the other hand, there were 6 BCL6 tive expression (Case 3) and more than cut-off values gain (10.5%), 10 (17.5%) BCL6 translocated cases and 3 (Case 14). There were three cases (Case 7, 12 and 15) (5.3%) BCL6 translocated and gain. While, FL had 3 showing triple gene aberrations of BCL2/BCL6/C-MYC (20%) BCL6 translocated cases and 1 (6.7%) BCL6 gain with at least 3 gain copies of chromosome or trisomy case. There was no BCL6 gene abnormalities found in 3. The three cases corresponding protein expression Other NHL subtypes. In the 57 DLBCL cases for presented more than 70% BCL2, more than 40% C-MYC aberrations, there were 5 (8.8%) C-MYC gain C-MYC and all were non-GCB subtypes. and 4 (7.0%) C-MYC translocated cases. While, FL had

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Figure 1. DLBCL case of GCB origin with double-hit C-MYC/BCL6 translocation (Case 6) at x63 magnification. White arrow shows the discrete red and green signals. (A) C-MYC translocation (B) BCL6 translocation.

Table 4. Immunohistochemical and genetic features of the 16 DLBCL and FL cases

No. Diagnosis BCL2 BCL6 C-MYC Ki67 IgH IHC FISH IHC FISH IHC FISH IHC FISH Case 1 FL positive T&G positive x x G (3- 4 cp) x x Case 2 FL positive T positive N x G (up to 3 cp) >80 T Case 3 FL positive T positive T x N 50 T Case 4 DLBCL >70% G (up to 3 cp) <50% G (3- 4 cp) <40% N >90 x Case 5 DLBCL >70% G (2- 3 cp) <50% T <40% N >90 N Case 6 DLBCL >70% N <50% T <40% T 40 T Case 7 DLBCL >70% G (3- 4 cp) >50% G (3- 4 cp) >40% G (3- 4 cp) 50 G (3- 4 cp) Case 8 DLBCL >70% G (up to 3 cp) ~50% x <40% T >80 x Case 9 DLBCL >70% G (2- 3 cp) >50% N >40% T ~50 N Case 10 DLBCL >70% G (3- 4 cp) ~50% T&G <40% N >95 N Case 11 DLBCL >70% G (3- 4 cp) >50% G (2- 3 cp) >40% N 90 N Case 12 DLBCL >70% G (4- 5 cp) >50% G (up to 3 cp) >40% G (up to 3 cp) ~100 N Case 13 DLBCL >70% G (4- 5 cp) >50% T&G >40% G (3- 4 cp) ~100 T&G Case 14 DLBCL >70% T >50% T <40% G (3- 4 cp) >90 T Case 15 DLBCL ~70% G (5- 6 cp) <50% G (up to 3 cp) >40% G (3- 4 cp) x T&G Case 16 DLBCL >70% G (3- 5 cp) ~50% T&G <40% N 80 N N, Normal; G, Gain copies; T, Translocation; T&G, Translocation & Gain copies; cp, copies; X, Missing/No tissue left

Table 5. Summarise of gene abnormalities among BCL2 / BCL6 / Comparing the genetic aberrations with the C-MYC / IgH in 16 cases of B-cell NHL protein expression of the 16 cases showed that all of N the 16 cases had positive protein expression for both Male (n=8), Female (n=8) BCL2 and BCL6. In the 13 cases of DLBCL, all of them Nodal (n=12), Extranodal (n=4) had BCL2 protein expression of more than 70%. 9 BCL2/BCL6 BCL2/BCL6 - (G) 2 DLBCL cases had BCL6 protein expression of more BCL2/BCL6 - (T) 1 than 50% and only 6 DLBCL cases had more than 40% BCL2 (G) / BCL6 (T) 1 C-MYC protein expression. In the FL cases, 2 had BCL2 (G) / BCL6 (T&G) 2 BCL2/C-MYC BCL2/C-MYC gene aberrations and the other had BCL2 (G) / C-MYC (T) 2 BCL2/BCL6 translocation. In the cases involving BCL2 (T) / C-MYC (G) 1 extranodal sites, all 4 cases had gain of BCL2 (T&G) / C-MYC (G) 1 at least 3 copies in BCL2. BCL2/BCL6 had the highest BCL6/C-MYC BCL6/C-MYC - (T) 1 gene aberrations combination (6 cases) compared to BCL2/ BCL6/C-MYC BCL2/C-MYC (4 cases) or BCL6/C-MYC (1 case). With BCL2/ BCL6/C-MYC - (G) 3 the exception of Case 1, 6 and 15, the cell proliferation BCL2/C-MYC (G) & BCL6 (T&G) 1 for the 16 cases showed at least 50% Ki67 index. BCL2/ BCL6 (T) & C-MYC (G) 1 G, Gain copies; T, Translocation; R&G, Translocation & Gain copies

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Discussion examined data using methods that eliminate most false-negative results by detecting BCL2 sequence In the present study, we reported the prevalence rearrangements outside of the MBR (major and genetic abnormalities of 81 cases of B-cell NHL breakpoints) and mcr (minor breakpoints) sites. In among Malaysian patients using interphase FISH. addition, BCL2 translocation is not sufficient to cause Our findings revealed there was a significant B-cells transformation as seen in BCL2 transgenic difference detected between the nodal and extranodal mice, which only develop lymphomas after secondary sites in BCL2, C-MYC and IgH. The findings coincide chromosome modifications and long latency period with a study [20] investigating on metaphase [31]. Marin et al. [32] and Tanaka et al. [33] have both comparative genomic hybridization (CGH) and their established in vivo that BCL2 deregulation caused by results showed nodal sites having more frequent translocation of BCL2 increases cell survival by amplifications than extranodal particularly of BCL2. preventing apoptosis, prompting the cell to acquire The differences could readily be explained by the secondary chromosomal aberrations. BCL2 gene alterations in the behaviour of the cell types from the aberrations, other than t(14;18)(q32;q21), such as sites they develop [21]. The findings connected 18q21 amplification or activation of the nuclear previous evidences [22,23] that high gene aberrations factor-kB pathway, has been suggested to be the main at site-specific lymphomas may increase the number responsible act for dysregulation of BCL2 protein of lymphocytes, hence higher chances of a malignant expression in the DLBCL non-GCB subtype [34]. clone developing. In addition, certain local factors It was also found that in our FISH analysis of 50 such as exposure to numerous types of antigens and DLBCL cases, the frequency of C-MYC aberrations of activation of the B-cell receptor (BcR) signalling 18% was quite similar to what have been reported in pathway are known to increase the risk of lymphomas Western populations [35]. C-MYC rearrangement at at nodal or extranodal sites [24]. This is important as band 8q24, is associated with a poor prognosis and extranodal lymphoma is distinctive from nodal in reported in approximately 15% of DLBCL cases [35]. numerous ways, such as in treatment strategies and However, supported data to incriminate geographical prognosis. Apart from that, it is shown that among or genetical factors between DLBCL in South East Asian population, extranodal sites such as Asian and in Western countries needs further gastrointestinal tract, nasal cavity and tonsils are the observation that can be confirmed in larger studies. commonest sites and have high incidence compared Deregulation of C-MYC has been implicated with to nodal sites [25]. Undoubtedly, there is more to be aggressive lymphomas and adverse prognosis in learned of DLBCL pathogenesis at distinct extranodal B-cell malignancies as it plays a role by promoting cell sites since there is also emerging cases of differences cyle progression and tumour proliferation in DLBCL. and similarities in somatic genetic abnormalities Using the same samples, we had identified the MYC particularly between DLBCL arising at different sites involvement in STAT3-mediated pathway in EBV [26,27]. However, there is little evidence in these data (+)-DLBCL cases [36]. Substantial evidence [37] of to suggest that aberrations in the nodal and both in vitro and in vivo models have provided that extranodal sites could have effects on the clinical MYC prompts many genes that involved in ribosome course and survival due to insufficient information of biogenesis including genes associated in glucose and the patients. glutamine metabolism to accommodate the growing BCL2 translocation was found in 40% and 32% of lymphoma cells. Translocation of MYC has been our 13 FL and 51 DLBCL cases respectively. The BCL2 described as the genetic hallmark and the driving translocation is the cytogenetic hallmark for for BL [38] as presented in our only BL case. approximately 90% of FL cases. Although the BL is a germinal center tumour harbouring somatic incidence of FL among Asian is considerably smaller hypermutation of the Ig genes and is characterized by compared to Caucasian, a Japanese study [28] found the presence MYC and IgH translocation. Although, high (81%) BCL2 translocated FL cases using FISH C-MYC is the defining feature to BL, it has also been analysis in contrast to our FL findings with only 40% recognized in other NHL B-cell lymphomas such as BCL2 translocation. Similar findings in the Western DLBCL and FL whereby MYC overexpression has countries, Germany [29] generated quite similar been associated in the transformation of FL [39]. results with only 53% of BCL2 translocation in early On the other hand, there was no significant stage FL cases. Apart from the reason that we had difference for BCL6 gene aberrations found between considerably low FL sample size, it was also the nodal and extranodal sites. BCL6 translocation suggested that the discrepancy between the findings and clinical features of B-cell NHL particularly could be due to technique differences as suggested by DLBCL has been the subject of controversy. Several a study [30] that investigated variations of the BCL2 studies [40,41] reported that BCL6 gene translocations translocation incidence in FL. Aster and Longtine [30]

http://www.jcancer.org Journal of Cancer 2020, Vol. 11 196 occured frequently in extranodal than in nodal sites. chains occurring only after heavy chain gene Subsequent studies by Offit et al. [42] found that rearrangement has occurred through allelic exclusion extranodal DLBCL patients had more frequent gene [53]. Under normal circumstances, only one of the two rearrangements than those found in the nodal sites of IgH alleles is functional due to its monoallelical nature DLBCL. However, contradicting report [43] which and to facilitate allelic exclusion, the other allele is also supported findings in this study found that there non-functional for several reasons, in this particular was no difference in the BCL6 translocation between case the non-functional allele is translocated but the DLBCL sites. encoded on a dysfunctional Ig chain therefore cannot Our DLBCL findings also revealed that BCL6 be assembled into a surface-expressed BcR or an had the highest gene translocation (40%) compared to [54]. Another reason IgH-specific gene BCL2 and C-MYC. The incidence of BCL6 aberrations could occur was due to the problematic translocation in our study supported the evidence nature of the FISH IgH probe whereby we used three that BCL6 gene translocation in B-Cell NHL times signal diameter for IgH image analysis constituted about 30-40% of all DLBCL [44,45]. In confirmation of translocation in our study. Due to the addition, 6% of our FL cases also had BCL6 gene unique mechanism of V(D)J recombination, the green aberrations. Although it is unusual, previous study signals will be weak due to deletion. Sometimes showed that 5-15% of FL patients had BCL6 following a translocation, a signal may be lost as the rearrangement that occurred in the both low-grade derivative partner chromosome is deleted or if the and high-grade FL [29, 46]. It was suggested that FL probe has been deleted by recombination. Hence, IgH cases with translocation other than BCL2 may have green probe may faintly cross hybridise with small unconventional molecular mechanisms or at least an regions located on 15q11.2 and 16p11.2 showing tiny alternate initiating event of FL [29]. About 13 out of 23 green dots accompanying the larger IgH green signals. cases of the BCL6 gene aberrations in this study With regards to double or triple aberrations involved IgH gene while the rest are with BCL2 and among the three genes, our study had one double-hit C-MYC, showing the diversity of BCL6 translocation lymphoma of BCL6/C-MYC found in DLBCL Case 6, partner as mentioned by Cattoretti et al. [47]. It is with both of the BCL6/C-MYC protein expression identified that non-IgH/BCL6 translocated cases are showing less than 50% and 40% respectively and Ki67 under the effect of the regulatory regions in BCL6 and of 40%. The BCL6 and C-MYC cut-off values were some of these partner genes expression have role(s) in carefully considered and adopted from previous the development of DLBCL [48,49]. BCL6 gene studies [55,56] investigating the C-MYC, BCL2 and rearrangement has been associated with poor BCL6 expression in DLBCL patients. In addition, our prognosis when a study [50] evaluating BCL6 study also found two cases harbouring both translocation using FISH analysis found that BCL2/BCL6 translocation at the nodal site. One of this translocation of BCL6 in DLBCL patients had BCL2/BCL6 double translocation were discovered in significantly lower overall survival than those low grade FL case which was in accordance with without translocation. Consistent to what has been previous studies [57-59] that reported the BCL2/BCL6 mentioned previously [51], we found significant translocation was predominantly low grade FL. higher BCL6 gene abnormalities in non-GCB subtype Although there are contrasting evidence with regards (38%) than in GCB subtype (2%) (p=0.03), suggesting to BCL2 and BCL6 rearrangement prognostic impact that the DLBCL GCB and non-GCB subtype have [50], consensus exist that C-MYC rearrangement is the separate molecular mechanisms of DLBCL worse prognostic marker for DLBCL patients treated pathogenesis responsible for the abnormal protein with either standard CHOP or R-CHOP[5]. expression. However, BCL6 gene aberrations were not Pfreundschuh [60] mentioned that, apart from correlated with high BCL6 expression level in our translocation, FISH failed to detect other gene study (p=1.00). Akyurek et al. [52] reported that there aberrations; however our study found other types of was no correlation between BCL6 rearrangement with C-MYC, BCL2 and BCL6 gene aberrations such as gain the level of BCL6 protein expression. However, this copies which was detected through FISH break-apart discrepancy can also arise due to the differences in the probe analyses. There were three DLBCL cases having staining and scoring methodologies, cut-off values, at least trisomy 3 of BCL2/BCL6/C-MYC and all were and sample populations. non-GCB subtypes. Asian and Western [61] studies There were also 8 cases with IgH only gene revealed that the ABC subtype of DLBCL has frequent aberrations which warrant further investigations. It is trisomy 3 especially involving chromosome 3 (BCL6) known that IgH is the first rearranged gene in B-cell and 18 (BCL2) as defined by profiling. development and an early marker of clonal B On the other hand, the small population of the GCB lymphoid processes, with light chain genes for κ and λ subtype in this study was found to be less compared

http://www.jcancer.org Journal of Cancer 2020, Vol. 11 197 to the non-GCB subtype, consistent with previous Competing Interests studies [62] on the prevalence of GCB-subtype on The authors have declared that no competing Asian patients. It was found that Asian DLBCL interest exists. patients had less frequent GCB subtype compared to Caucasian patients, a difference that may be References attributed to ethnic factors. 1. Baris D, Zahm SH. Epidemiology of lymphomas. Curr Opin Oncol. 2000; 12(5): In summary, few interesting patterns emerged 383-94. 2. Devesa SS, Fears T. Non-Hodgkin's lymphoma time trends: United States and that should be interpreted with caution. Our data international data. Cancer Res. 1992; 52(19 Suppl): 5432s-40s. suggest that there is a significant difference between 3. Müller AM, Ihorst G, Mertelsmann R, Engelhardt M. Epidemiology of non-Hodgkin’s lymphoma (NHL): trends, geographic distribution, and the sites of lymphoma hence suggest that lymphoma etiology. 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